Expanding the genetic code of Escherichia coli with phosphotyrosine

Abstract

Protein phosphorylation is one of the most important post-translational modifications in nature. However, the site-specific incorporation of O-phosphotyrosine into proteins in vivo has not yet been reported. Endogenous phosphatases present in cells can dephosphorylate phosphotyrosine as a free amino acid or as a protein residue. Therefore, we deleted the genes of five phosphatases from the genome of Escherichia coli with the aim of stabilizing phosphotyrosine. Together with an engineered aminoacyl-tRNA synthetase (derived from Methanocaldococcus jannaschii tyrosyl-tRNA synthetase) and an elongation factor Tu variant, we were able to co-translationally incorporate O-phosphotyrosine into the super-folder green fluorescent protein at a desired position in vivo. This system will facilitate future studies of tyrosine phosphorylation.

Translation system engineering in Escherichia coli enhances non‐canonical amino acid incorporation into proteins

“…One of our goals in this manuscript was to explore the possibility that EF-Tu might be a more general target for translation system engineering, especially given that interactions between EF-Tu and aminoacyl-tRNAs are tuned to perfectly match natural amino acids with cognate tRNAs (Schrader et al, 2009). Previous reports suggest that EF-Tu engineering can be beneficial for bulky amino acids (Doi et al, 2007) and charged amino acids as shown for phosphoserine (Park et al, 2011) and phosphotyrosine (Fan et al, 2016). Our results suggest that, it might be a more general target, although additional studies are needed to understand the generality of evolving EF-Tu alongside aaRSs to facilitate integration of a wide range of ncAA targets.…”

“…Consequently, ncAA-tRNA substrates may not bind EF-Tu efficiently because it is a non-native substrate, preventing efficient delivery to the ribosome. While still a subject of debate for ncAA incorporation efforts (Rogerson et al, 2015), EF-Tu may thus require engineering to allow for the efficient incorporation of a given ncAA (Fan et al, 2016; Park et al, 2011). Beyond o-aaRSs, o-tRNAs, and EF-Tu, ribosomes must be able to accommodate the ncAA and in some instances ribosome engineering may be necessary.…”

“…This is useful for expanding ncAA diversity without the need for further evolution, but hinders incorporation of multiple distinct ncAAs by existing o-aaRSs (Miyake-Stoner et al, 2010; Wan et al, 2014; Wang et al, 2012; Young et al, 2011). Native EF-Tu also shows limited capacity for incorporation of bulky or charged ncAAs (Fan et al, 2016; Park et al, 2011), and could be the target of engineering efforts if thermodynamic interactions limit delivery of aminoacyl-tRNA substrates (Wang et al, 2016). Further, the presence of release factor 1 (RF1) can cause early termination of proteins when using amber suppression technology because it competes for the UAG codon (Hong et al, 2014b; Johnson et al, 2011).…”

“…Second, efforts to suppress or delete RF1 have removed the competition with the ncAA-o-tRNA species at the UAG codon to increase incorporation efficiencies (Johnson et al, 2011; Lajoie et al, 2013; Loscha et al, 2012; Mukai et al, 2010; Ohtake et al, 2012). Third, efforts to further engineer individual OTS components have led to improved ncAA aminoacylation efficiencies and increased overall yields of modified proteins, for example, o-tRNA (Chatterjee et al, 2012; Young et al, 2010), o-aaRS (Amiram et al, 2015; Chatterjee et al, 2012; Liu et al, 1997), EF-Tu (Doi et al, 2007; Fan et al, 2016; Park et al, 2011; Wang et al, 2015), and ribosome (Barrett and Chin, 2010; Neumann et al, 2010; Rackham and Chin, 2005; Wang et al, 2007). …”

Rewiring protein synthesis: From natural to synthetic amino acids

“…Similar EF-Tu engineering has been conducted to effectively insert selenocysteine into proteins in a SECIS-independent manner [63]. Not surprisingly, these laboratory-evolved EF-Tu variants contain mutations of negatively-charged or neutral residues to positively-charged arginines in the amino acid binding pocket [62–64]. In addition to EF-Tu engineering, tRNA optimization has also been successfully performed to enhance ribosomal incorporation of long-chain amino acids [65].…”